微泡增强的超声空化阻断正常肝血流的机制研究

Our previous study found that microbubble-enhanced ultrasound had a fast and significantly haemostatic efficiency for liver haemorrhage after trauma, the main mechanism of blocked regional blood perfusion was extensive swelling of hepatocytes, which squeeze and shrink the size of the sinusoids and formed a joint compression on the regional liver circulation, but the exact mechanism of liver cells swelling is still unknown. We speculated it was mainly because the sonoporation could increase the cell permeability by shockwaves and microjets, affect osmotic pressure balance between internal and external cells, and finally result in cell swelling by taking up more ions and water into the cell. Besides, up-regulation of the aquaporin-9 (AQP9) expression and suppression of the Na-K-ATPase might also be one of the factors contributed to liver cells swelling. On the basis of previous studies, this research would observe the changes of cell membrane integrity and permeability, AQP9 expression level, Na-K-ATPase activity, and intracellular ions (Na+, K+, Ca2+) concentration, which are generated by microbubble-enhanced ultrasound. The purposes of this studies are to investigate the relationship between sonoporation, AQP9, Na-K-ATPase and liver cell swelling, explore the exact mechanism of regional blood perfusion blocked by ultrasound, furthermore, it is hoped that we can control the degree of liver cell swelling by adjusting the parameters of ultrasound and the dosis of microbubbles, and finally, pave the way for controlled treatment using microbubble-enhanced ultrasound, such as liver trauma haemorrhage or other therapic applications on livers.

前期实验发现，微泡增强的超声空化效应应用于肝脏创伤出血时有显著快速的止血作用，其局部血流阻断机制主要是广泛的肝细胞肿胀暂时性压迫肝窦，但肝细胞肿胀的具体机制不明。推测主要是由于超声声孔效应导致肝细胞膜通透性增加，影响了细胞内外渗透压平衡造成的水钠储留，水通道蛋白9表达上调和钠钾ATP酶活性降低可能也是其中的影响因素。本研究拟通过观察微泡联合超声辐照时肝细胞膜表面完整性和通透性的变化，检测其膜上水通道蛋白9表达水平、钠钾ATP酶活性及细胞内Na+、K+和Ca2+ 浓度的变化，论证声孔效应、水通道蛋白9和钠钾ATP酶在肝细胞肿胀中发挥重要作用的假说，探讨微泡增强的超声空化用于肝脏局部血流阻断的具体机制，并希望通过调整超声治疗参数控制肝细胞肿胀的程度，为以后微泡增强的超声空化可控的用于肝脏创伤出血或其他治疗方面的应用奠定基础。

微泡增强的超声空化效应应用于肝脏创伤出血时有显著快速的止血作用，其局部血流阻断机制主要是广泛的肝细胞肿胀暂时性压迫肝窦，但肝细胞肿胀的具体机制仍不明确。本课题采用析因设计法探究了两个重要超声空化参数——声空化辐照时间及峰值负压的不同组合对组织血流阻断效应的不同影响作用，从而筛选出了稳定有效的空化辐照参数，并在此基础上检测了组织水肿相关分子——钠钾ATP酶（ATPase）及水通道蛋白4（AQP4）表达量的改变，从而评价空化治疗后组织水肿与超声空化辐照参数之间的关系，以指导超声治疗参数的选择。研究结果发现，在声辐照时间为1分钟，5分钟，10分钟，峰值负压为1.0MPa，1.5MPa，2.0MPa的参数区域内，仅1.0MPa辐照1分钟即可实现显著有效的肝脏血流阻断，随着峰值负压的升高，辐照区域血流流速显著减缓，但辐照时间的延长无此效应。而ATPase及AQP4表达量随辐照时间的延长及峰值声压的增高均显著增加，证实了高能量超声空化可造成肝脏组织水肿的加重。该研究工作不仅再次证实了低能量超声辐照联合微泡可有效实现肝脏的止血，更通过系统性研究方法探讨了超声辐照参数的调整对止血效果及组织损伤的影响，有助于指导在损伤最小化的情况下有效实现血流灌注的阻断。以上研究结果对低能量超声辐照这种快速、便捷的治疗方式在肝脏等内脏出血紧急救治中的临床推广具有重要的应用指导意义。